[CANCER RESEARCH 54, 915-919. February 15, 1994[
Reversible Conjugation of Ethacrynic Acid with Glutathione and Human
Glutathione S-Transferase PI-1
J. H . T. M . P l o e m e n , z A . V a n S c h a n k e , B. V a n O m m e n ,
a n d P. J. V a n B l a d e r e n
Department of Biological Toxicology, TNO Toxicology and Nutrition Institute, P O. Box 360, 3700 AI Zeist, the Netherlands
ABSTRACT
The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an a,~-unsaturated ketone, with glutathione and glutathione S-transferase PI-1 (GST PI-I) has been studied. When the glutathione conjugate of EA was incubated with a 5-fold molar excess of Nacetyi-L-cysteine or GST PI-I, a time-dependent transfer of EA to
N-acetyI-L-cysteine or GST PI-1 was observed. With increasing pH, the
pseudo first order rate constants of transfer of EA to N-acetyl-L-cysteine
increased from 0.010 h -~ (pH 6.4) to 0.040 h -I (pH 7.4) and 0.076 h -~
(pH 8.4).
From the fact that preincubation of GST PI-I with l-chioro-2,4-dinitrobenzene reduced the incorporation of p4C]EAfrom 0.94 _+0.21 (SD) to
0.16 + 0.02 mol EA/moi subunit and from automated Edman degradation
of the major radioactive peptide isolated after pepsin digestion of the
[~4C]EA-iabeled enzyme, it was concluded that the reaction of EA takes
place with cysteine 47 of GST PI-1.
When GST PI-1 was inactivated with a 5-fold molar excess of EA,
adding an excess of glutathione resulted in full restoration of the catalytic
activity in about 120 h.
These findings may have several implications. Under normal physiological conditions the inhibition of GST PI-1 by covalent binding of EA
would be reversed by glutathione, leaving reversible inhibition by the
glutathione conjugate of EA and by EA itself as the main mechanism of
inhibition; however, when glutathione levels are low the covalent inhibition might be predominant, resulting in a completely different time course
for the inhibition.
INTRODUCTION
Conjugation with the tripeptide glutathione is considered to be an
important detoxification reaction for electrophilic xenobiotics, including numerous cytostatic agents. In general this reaction is catalyzed by
GST 3 (1-3). Most GSTs occur in cytosol and belong to one of four
multigene families, termed alpha, mu, pi, and theta (1, 4).
The effectiveness of many clinically useful anticancer drugs is
severely limited by drug resistance. Several findings suggest the involvement of GST, besides other mechanisms, especially with respect
to resistance to alkylating agents such as chlorambucil and melphalan
(5-7). These findings include GST overexpression of especially the pi
class in tumors (8, 9), the direct conjugation of alkylating agents by
GST (10, 11), and the overexpression of GST in yeast and mammalian
cell lines by genetic engineering which confers to these organisms
resistance to alkylating agents (6, 12). A promising strategy to overcome this alkylator resistance phenotype may be based on inhibition
of GST.
The diuretic drug ethacrynic acid, an a,13-unsaturated ketone, is a
potent reversible inhibitor of GST isoenzymes (13-15) and has been
used to study the role of GST in drug resistance in vitro, using cell
lines (16), and colon tumor xenografts (17) and in a phase I clinical
study with the cytostatic agent thiotepa (18). Moreover, a concentration-dependent inhibition by ethacrynic acid of the enzyme-catalyzed
conjugation of glutathione with the clinically important alkylating
agent chlorambucil has been reported (11). The reversible inhibition
would further be enhanced by the formation of the glutathione conjugate of ethacrynic acid, which is an even stronger inhibitor for all
GSTs but the pi class (14). For both human and rat GST of the pi class,
covalent modification of GST concomitant with an irreversible loss of
activity could be achieved using slightly more drastic incubation
conditions (19). Conjugation with glutathione does not always lead
to the detoxification of electrophilic xenobiotics (20). In addition to
glutathione conjugates that are reactive by themselves, other types of
glutathione conjugates may undergo further metabolism to a reactive
species (20). A special case involves glutathione conjugates that exert
their toxic effects through release of reactive species; the glutathione
conjugates serve as transport and targeting agents. This situation occurs when the glutathione conjugation reaction is reversible, as found,
e.g., for some methyl isocyanates (21) and isothiocyanates (22). The
Michael addition of glutathione with c~,13-unsaturated aldehydes and
ketones is another well known reversible reaction (23). A reversible
Michael reaction has, e.g., been shown to be involved in the covalent
of an a,/3-unsaturated ketone metabolite of the veterinary drug furazolidone to glutathione and protein (24).
Since ethacrynic acid also contains an o~,/3-unsaturated ketone
moiety, the present study was designed to investigate the reversible
covalent interaction of ethacrynic acid with glutathione as well as with
GST PI-1. The interaction of ethacrynic acid with this enzyme was
included, since the inactivation of the GST of the pi class in several
cases has been shown to be the result of the modification of a
highly reactive cysteine residue (25, 26) and since pi class, next
to alpha, is one of the primary GST classes that are involved in drug
resistance (5).
M A T E R I A L S AND M E T H O D S
Received9/20/93; accepted 12/16/93.
The costs of publicationof this article were defrayedin part by the paymentof page
charges. This article must thereforebe hereby markedadvertisement in accordancewith
18 U.S.C. Section 1734 solely to indicatethis fact.
I This investigationwas supported by the NWO-Foundationfor Medical and Health
Research MEDIGON (Grant 900-521-124).
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: GST, glutathioneS-transferase; CDNB, 1-chloro-2,4dinitrobenzene;HPLC, high-performanceliquidchromatography;RP, reversephase; dd,
doublet of doublets; d, doublet;s, singlet; m, multiplet;dm, doubletof multiplets.
Chemicals and Enzymes. Ethacrynic acid [2,3-dichloro-4-(2-methylene1-oxobutyl)phenoxy]acetic acid, S-hexylglutathione, N-acetyl-L-cysteine, and
Tris were from Sigma Chemical Co., St. Louis, MO. Epoxy-activated Sepharose 6B was purchased from Pharmacia, Uppsala, Sweden. [14C]Ethacrynic
acid (15 mCi/mmol) was purchased from Amersham, Buckinghamshire,
United Kingdom. Trifluoroacetic acid was from J. T. Baker Chemical Co., Inc.,
Philipsburg, NJ. Pepsin (from porcine gastric mucosa) was obtained from
Boehringer Mannheim, Mannheim, Germany.
The radioactive conjugate of ethacrynic acid was prepared by adding 6
lxmol of glutathione in 180/xl of 0. ! Mpotassium phosphate buffer, pH 8, with
50% ethanol, to 1.3/.tmol of [14C]ethacrynic acid. After overnight incubation,
the glutathione conjugate of ethacrynic acid was purified by preparative RPHPLC using Zorbax ODS (Dupont; 21.2 * 250 mm), eluted at a flow rate of
4 ml/min with 0.01% formic acid (solvent I) and methanol (solvent II), with a
linear gradient of 40-100% solvent II in 60 min (k' = 2.0 and 3.1 for the
conjugate and ethacrynic acid, respectively). About 70% conversion of ethacrynic acid to the glutathione conjugate was obtained. Methanol was removed
under N2, after which a stock solution (of 136/xM) of the glutathione conjugate
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RETRO MICHAEL CLEAVAGE OF GLUTATHIONE CONJUGATES
was stored at -30~ A product of 95+% purity was obtained as judged with
RP-HPLC analysis, with a retention time identical to that of the nonradioactive
conjugate (14).
The N-acetyl-L-cysteine conjugate of ethacrynic acid was prepared, in analogy to the synthesis of the glutathione conjugate (14). The 1H nuclear magnetic
resonance (400 MHz, D20) spectra of the N-acetyl-L-cysteine conjugate are
consistent with the expected structure (19), with the following information for
the ethacrynic acid part of 8 7.81/7.79 (dd, 1H, J = 8.8 Hz), 8 7.23 (d, 1H,
J = 8.8 Hz), 4.99 (s, 2H), 8 3.8 (m, 1H), 8 1.95/1.81 (dm, 2H), 8 1.1 (m, 3H);
and for the N-acetyl-L-cysteine part of 8 2.19 (s, 3H, -CH3). The proton signals
of cys a overlap with D20 (6 = 4.7), while the signals of cys/3 and the protons
next to the sulfur atom (of the ethacrynic acid moiety) were found in the region
of 8 2.9-3.1, as a complicated multiplet pattern.
GST PI-1 was purified from human placenta as described (19). Protein was
determined by the method of Lowry et al. (27), using bovine serum albumin as
standard.
Incubations. The glutathione conjugate of ethacrynic acid (0.5 mM) was
incubated at 20~ with N-acetyl-L-cysteine (2.5 mrs) in 0.4 ml of 0.1 M
potassium phosphate buffer with 0.1 mM EDTA at three pH levels (6.4, 7.4, and
8.4). For each pH, 22 independent samples were prepared. At each time point,
20/xl were injected on RP-HPLC, using a Zorbax ODS (250 * 4.6 mm; LC
Service, the Netherlands) column and eluted at a flow rate of 1 ml/min with
0.1% trifluoroacetic acid in deionized water (solvent A) and in methanol
(solvent B), with a linear gradient of 30-95% solvent B in 18 min, followed by
2 min at 95% solvent B (k' = 4.5, 5.1, and 6.0 for the glutathione conjugate,
N-acetyl-L-cysteine conjugate, and ethacrynic acid, respectively). Peak areas at
270 nm were integrated with Nelson Analytical Model 2600 Chromatography
Software. EDTA was added to the incubations to prevent the trapping agent
N-acetyl-L-cysteine and the free glutathione from oxidation to their disulfides.
Since no free ethacrynic acid could be detected, it is concluded that EDTA
protected sufficiently against oxidation.
Covalent binding of [14C]ethacrynic acid was studied in a volume of 75/xl
0.1 Mpotassium phosphate buffer (pH 7.4) with 0.1 mM EDTA (buffer A), after
preincubation of 25 tXMGST PI-1 for 75 min at room temperature with (n =
3) or without (n = 2) 1 mM CDNB, whereafter the enzyme was incubated for
110 min with [14C]ethacrynic acid (final concentration, 100 /XM). Enzymebound ethacrynic acid was separated from ethacrynic acid by RP-HPLC
(Vydac TP5 column; Chrompack, the Netherlands; 200 * 3 mm). Elution was
performed with a flow of 0.6 ml/min, with solvent A (see above) and 0.1%
trifluoroacetic acid in acetonitrile (solvent C), with a linear gradient of 30--60%
solvent C in 30 min (k' = 4.0, and 6.3 for ethacrynic acid and enzyme with
bound ethacrynic acid, respectively). UV detection (at 214 nm) was used to
identify the enzyme peak, while simultaneously the radioactivity was measured
using an on-line radiochemical detector.
The [14C]ethacrynic acid-labeled GST PI-1 (0.25 mg) was digested with
pepsin [enzyme/protein ratio, 1/20 (w/w)] in 0.05 MTris/H3PO4 (pH 1.8) for 18
h at 37~ The pepsin-peptide mixture was purified on a RP column (Cl8,
Vydac Protein & Peptide USA; 250 * 4.6 mm) and eluted with solvents A and
C (see above), 5 min isocratically at 100% solvent A, followed by a linear
gradient from 0--60% solvent C in 70 min (flow rate, 1 ml/min). The main
radioactive peak was repeatedly purified on the same column. The peptide was
degraded using automated Edman degradation on an Applied Biosystems
Model 475 peptide sequencer on-line connected to a Model 120A PTH
analyzer.
The catalytic activity of GST PI-1, inactivated with ethacrynic acid, was
monitored after the addition of glutathione. GST PI-1 (1/XM) was preincubated
in buffer A (see above) with or without 10/xu ethacrynic acid (final volume,
200/xl), after which glutathione was added (final concentrations, 0, 10, 100,
and 1000 /XM).These incubations were performed in triplicate, at room temperature. At various time points, 20-pmol enzyme samples were transferred to
cuvetes, after which the catalytic activity towards CDNB was measured (28).
A time series was stopped when the remaining catalytic activity in the corresponding blank incubation was less than 70%.
To study the interaction of GST PI-1 with the glutathione conjugate of
[14C]ethacrynic acid, seven independent samples of 10 p.M GST PI-1 were
incubated at room temperature with 2/XM radioactive glutathione conjugate in
buffer A (see above) (final volume, 50 /xl). To separate the glutathione con-
jugate and free ethacrynic acid from the enzyme with bound ethacrynic acid,
30/~1 were injected on the Vydac TP5 column (see above).
RESULTS
The occurrence of the retro Michael cleavage of the glutathione
conjugate of ethacrynic acid was studied by incubation of the glutathione conjugate of ethacrynic acid with an excess of N-acetyl-Lcysteine (Fig. 1). The transfer of the ethacrynic acid moiety to Nacetyl-L-cysteine was followed with time by quantification of the
glutathione and N-acetyl-L-cysteine conjugates of ethacrynic acid on
RP-HPLC. The rate of transfer increased with increasing pH, with
pseudo first order rates of 0.010, 0.040, and 0.076 h -1 for pH 6.4, 7.4,
and 8.4, respectively. After 180 h of incubation at pH 8.4, an equilibrium was reached between the glutathione and the N-acetyl-L-cysteine conjugate, suggesting that the distribution of ethacrynic acid
over glutathione and N-acetyl-L-cysteine is mainly determined by their
relative concentrations.
Retro Michael cleavage can also occur with GST P l - l - b o u n d ethacrynic acid, if the assumption is right that ethacrynic acid reacts with
a cysteine residue of GST PI-1 (19). In order to check this assumption, the incorporation of [14C]ethacrynic acid in GST PI-1 after
preincubation with CDNB was studied, which is known to inactivate
GST PI-1 by modification of cysteine 47 (25). Identical to an earlier
study (19), 0.94 ___ 0.21 nmol label/nmol GST PI-1 could be incorporated in blanc incubations. As expected, CDNB protects against
incorporation of ethacrynic acid; 0.16 _+ 0.02 (SD) nmol label/nmol
GST PI-1 could be incorporated after preincubation with CDNB,
supporting the hypothesis that ethacrynic acid reacts with cysteine 47
of GST PI-1. In order to definitely identify the amino acid involved
in the reaction, the GST PI-1 with bound ethacrynic acid was digested
with pepsin and the resulting peptides were separated on HPLC. A
main radioactive peak was identified, which contained >80% of the
radioactivity, eluting at 45 min (Fig. 2). The amino acid sequence of
this peptide indicated that it spans residues 44--46 in the primary
amino acid sequence of GST PI-1 (Lys-Ala-Ser) (29-31), while an
unknown residue was observed in the 4th cycle (presumably the
cysteine-ethacrynic acid adduct). Thus it was again concluded that
cysteine 47 is the main target site.
1.00
0.60
= ~
~
/
o/
0 . 6 0
0.40
0.20
,/x.,,AA.6,~/X/~
0.00 =
0
,
,
,
-"
J
I
50
=
i
i
i
I
i
1 O0
time
i
,
i
I
i
150
i
i
i
200
(h)
Fig. 1. Transfer of ethacrynicacid from its glutathione conjugate to N-acetyI-L-cysteine. The glutathioneconjugateof ethacrynicacid (0.5 mM) was incubated at 20~ with a
5-fold excess of N-acetyl-L-cysteine.The transfer of the ethacrynic acid moiety to Nacetyl-L-cysteinewas followedwith time by quantificationof the conjugateson RP-HPLC
by integration of the peak areas at 270 nm. The reaction was studied at three pH levels,
6.4 (&, &), 7.4 (0, O), and 8.4 01, []). Open symbols, newly formedN-acetyl-L-cysteine
conjugates; closed symbols, the glutathione conjugate of ethacrynicacid.
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RErFRO
MICHAEL
CLEAVAGE
OF
GI.UTATHIONE
CONJUGATES
9000
8000
7000
6000
5000
Fig. 2. HPLC analysis of the pepsin digest of
ethacrynic acid-labeled GST PI-1. From l-min
fractions, samples(40/xl) were screenedfor radioactivity. Inset, peptides monitored from 0 to 75
min at 214 rim; peak at 44.8 min indicated (full
scale, 0.3 absorbanceunit full scale).
4000
3000
2000
1000
.
.
.
.
.
.
.
0
.
.
.
.
.
.
.
.
.
10
.
.
.
.
.
.
.
.
.
.
.
.
20
30
40
50
60
70
time (min)
Then, GST PI-1 (1 /~M) was incubated with ethacrynic acid (10
/XM), resulting in 90% loss of activity towards CDNB, and glutathione
was added. The catalytic activity toward CDNB was measured over a
120-h period (Fig. 3). Full restoration of the catalytic activity occurs
60
oot
ii
>,
~
>
. m
40
c
DISCUSSION
C
E
|
'--
with 0.1 and 1 mM glutathione (Fig. 3, inset). The 10 /XM incubation
initially shows partial restoration of catalytic activity, but after prolonged incubation loss of catalytic activity is observed, probably due
to oxidation. This is also observed in the corresponding control incubation (a 30% loss of activity in about 30 h; result not shown). Without
a trapping agent for free ethacrynic acid, no restoration of activity was
observed (Fig. 3).
In order to investigate whether retro Michael cleavage of the glutathione conjugate of ethacrynic acid concomitant with incorporation
of ethacrynic acid in GST PI-1 occurs, a 5-fold molar excess of the
enzyme was incubated with the glutathione conjugate of [taC]ethacrynic acid. A time-dependent increase of enzyme-bound label was
observed (Fig. 4), in accordance with the reversible nature of the
reactions.
20
\
A'A-"-,
0
A'A
,
A
,
,
I
J
i
L
10
A
t
20
t i m e (h)
,
i
i
I
30
a,/3-unsaturated aldehydes and ketones have long been known to
form conjugates with glutathione, both spontaneously and enzyme
catalyzed (32). The extent to which the enzyme plays a role differs
widely among members of this class of compounds (33). The chemical
reaction involved in the conjugation of ethacrynic acid and structurally related compounds, a Michael addition, is reversible. In the present study, it was shown that this retro Michael cleavage of ethacrynic
acid and glutathione indeed occurs. Thus, ethacrynic acid may be
transferred from one low molecular weight compound to another or to
reactive and accessible cysteines in proteins, e.g., cysteine 47 of GST
PI-1 as observed. Transfer from the glutathione conjugate of ethacrynic acid might take place in one of two ways, namely a release
from the glutathione conjugate in the active site of the enzyme and/or
reconjugation of released ethacrynic acid from the free glutathione
conjugate in the incubation medium, followed by binding to the enzyme. This transfer phenomenon is probably a common feature of
aJ3-unsaturated aldehydes and ketones; transport via a thiol conjugate
and subsequent regeneration of the reactive agent thus may be involved in the biological activity of such adducts (23).
Fig. 3. Restorationof the catalytic activityof ethacrynicacid-inactivatedhuman GST
PI-1 by incubationwithglutathione.GST PI-1 (1/XM)was incubatedwith ethacrynicacid
(10/XM),resultingin 90% lossof catalyticactivitytowardsCDNB. Glutathionewas added
[0 (A), 0.01 (O), 0.1 (E]), and 1 (0) raM]. The catalytic activity toward CDNB was
measured over a 120-h period and expressedas a percentageof control incubationswith
non-ethacrynicacid-modifiedenzyme(note: incubationswere stopped when the remaining activity in the correspondingcontrol was less than 70%). Inset, effect of prolonged
incubation. Individualpoints are the average of three measurements,with coefficientsof
variation less than 15%.
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RETRO MICHAEL CLEAVAGE OF GLUTATHIONE CONJUGATES
Recently, it was shown that chronic exposure of human colon
carcinoma cells to ethacrynic acid led to a 2-3-fold increase of GST
PI-1 activity, by an induction of the enzyme at the transcriptional level
(37). This phenomenon has been proposed by Talalay et al. (38) to be
a general one; compounds that contain a Michael acceptor or from
which a Michael acceptor can be formed during metabolism are usually inducing agents for GST. The contrasting effects observed for
Michael acceptors, i.e., inhibition of GST by covalent modification
and induction of GST, deserve further attention.
Furthermore, some otj3-unsaturated aldehydes are established inhibitors of growth. In the case of 4-hydroxynonenal and related compounds it was shown that inhibition of DNA synthesis was involved,
presumably as a result of a reaction with a functional sulfhydryl group
of DNA polymerase (23). More recently, another type of growth
inhibition has been reported, which involves the ot,/3-unsaturated
ketone A~2 prostaglandin J2, a cyclopentenone prostaglandin which
readily forms glutathione conjugates (39). These conjugations should
in principle also be able to undergo retro Michael cleavage to reform
the parent compounds. Interestingly, it has been shown that ethacrynic
acid, along with other inhibitors of GST, also has antiproliferative
effects on cell lines (40), which seem to be reversible.
100
80
.~ -60
~
40
o
20
o
=,.~o/
0
5
10
15
20
25
time (h)
Fig. 4. Interaction of GST PI-1 with the glutathione conjugate of [14C]ethacrynic acid.
GST PI-1 (10/.ZM) was incubated with a 2 I~M concentration of the glutathione conjugate,
JaC-labeled in the ethacrynic acid moiety. To separate the glutathione conjugate and free
ethacrynic acid from the enzyme-bound fraction, a RP-HPLC method was used as described under "Materials and Methods." The transfer of the ethacrynic acid moiety is
expressed as a percentage of total 14C label.
REFERENCES
The nature of the inhibition of GST by ethacrynic acid has been
studied in detail, since it was reported that ethacrynic acid in vivo
bound covalently to rat GST 3-4 (34). Previously we showed that
ethacrynic acid and its glutathione conjugate were potent reversible
inhibitors of GST isoenzymes, with 50% inhibitory values in the range
of 1-10 /ZM (14). This reversible inhibition was suggested to be the
predominant inhibitory mechanism in vivo, since the incorporation of
ethacrynic acid in GST 3-4 did not appear to inactivate the enzyme
(14, 19). In the case of GST PI-1, it was shown recently that the
mechanisms of the reversible inhibition by ethacrynic acid and its
glutathione conjugate were distinct (noncompetitive and competitive,
respectively) (35). In the alpha and mu class the conjugate of ethacrynic acid was a more potent reversible inhibitor than the parent
compound, while for the pi class some conflicting results have been
reported (14, 35).
GSTs of the pi class are inhibited by covalent binding of o~,lBunsaturated aldehydes and ketones; acrolein, a toxic aldehyde that
occurs as an environmental pollutant (36), and also ethacrynic acid
(19), specifically inactivated GST of the pi class. It is now clear that
the inhibition is only transitory, since the chemical reaction is reversible. Full restoration of the catalytic activity can be achieved by
prolonged incubation with an excess of glutathione. In an earlier
study, only marginal inactivation of GST 7-7 by ethacrynic acid was
observed using overnight dialysis experiments (14). Presumably the
reversibility of the chemical reaction contributed to the failure of this
dialysis experiment to detect time-dependent inhibition of GST PI-1
by ethacrynic acid.
The implications of the present findings may be several. Firstly, this
mode of action, reversible covalent binding to GST PI-1 and glutathione, may have some significance for the use of ethacrynic acid as
an in vivo inhibitor for GST PI-1 in drug resistance. Under normal
physiological conditions [glutathione concentration 1-10 mM (1)],
glutathione may be expected to reverse any covalent binding of ethacrynic acid to GST PI-1, and the inhibition of GST would occur only
reversibly, through the glutathione conjugate of ethacrynic acid and of
ethacrynic acid itself. However, in those cells with high levels of GST
PI-1 and/or low levels of glutathione, covalent inhibition of GST PI-1
might be predominant. The time course of inhibition would be completely different in these two cases.
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Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer
Research.
Reversible Conjugation of Ethacrynic Acid with Glutathione
and Human Glutathione S-Transferase P1-1
J. H. T. M. Ploemen, A. Van Schanke, B. Van Ommen, et al.
Cancer Res 1994;54:915-919.
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